During this past year, we have been working on several projects which relate to understanding the role of modified nucleotides in mammalian tRNA. We have developed rapid, sensitive tRNA sequence analysis techniques and applied them to the elucidation of cytoplasmic and mitochondrial tRNA sequences. We have recently reported the complete nucleotide sequence of several human gly-tRNAs and have also recently completed the nucleotide sequences of asn-tRNAs which have been isolated from several different mammals. The results of these experiments confirm our earlier hypothesis that the primary structure of mammalian cytoplasmic tRNAs is conserved throughout higher eukaryotes, although Walker 256 mammary tumor tRNA lacks to G to Q post-transcriptional modification. We have also investigated two mammalian in vitro cell free protein synthesizing systems and have shown that the rT effect we observed in the poly U-directed system can also be observed with a natural m-RNA (i.e. hemoglobin). During this next year, we will extend these studies by investigating the effect of U to rT conversion on the kinetics of the translocation step in protein synthesis, and also study the efficiency of various tRNAs which show other post-transcriptional modification differences to change the rate of protein synthesis.